The present invention relates to a quantitative assay for polysaccharides, which can be used for the rapid identification of novel antimicrobial agents.
The natural tendency of a microorganism to mutate its genome in order to achieve antibiotic resistance provides an ongoing need for the identification of new antimicrobial agents. One class of antibacterial agents acts by inhibiting synthesis of the cell wall of the targeted microorganism. Examples of antimicrobial agents that act in this manner are penicillin and nacillin, a semi-synthetic version of penicillin. The ability of a compound to interfere with the synthesis of a cell wall in vivo can be estimated in an in vitro assay of polysaccharide levels following cell wall synthesis in vitro in the presence of the compound. Because the major structural polysaccharides in the cell wall of fungi are .beta.-glucan and chitin, compounds that interfere with the synthesis of these polymers may have utility as antifungal agents.
Many of the previously described methods for measuring polysaccharide levels in a sample involve the transfer of a radioactively labeled monosaccharide from an activated sugar donor to a specific saccharide acceptor. For example, the level of chitin synthesized in a reaction can be determined by measuring incorporation of radiolabeled GlcNAc from Uridine-Diphosphate GlcNAc into the growing chitin chain. After physical isolation of the generated chitin, one can quantitate the amount of chitin by taking into account the specific activity of the radiolabeled sugar donor.